Cytochrome P450-cholesterol side chain cleavage (P450-scc) catalyzes the NADPH and oxygen-dependent conversion of cholesterol to pregnenolone. The reaction is believed to be the rate-determining step for flux through the steroidogenic pathways and is the main point at which adrenocorticotropic hormone regulates steroid hormone biosynthesis. Our objectives are 1) to provide a thermodynamic and kinetic characterization of individual steps in the mono-oxygenase reaction cycle, and 2) to assess which specific steps within the cycle are rate-limiting under different conditions. We plan to use purified P450-scc as well as submitochondrial particle preparations from the adrenal cortex to investigate (1) redox potentials and effects of interaction with adrenodoxin, (2) equilibrium affinities of the enzyme for steroid substrates, the product pregnenolone, oxygen and carbon monoxide, and (3) the kinetics of the electron transfer reaction and the ligand binding and release steps.